In extensive studies, we found that the Eda gene (ZO1 AG000643-10 LG) is deeply involved in the initiation and specification of several skin appendages, including hair follicles and sweat glands -- both in humans and in mice defective in the cognate Eda gene. However, we found that sebaceous glands develop as a further appendage on hair follicles, form independently of the Eda pathway;but they become larger in response to Eda action. This has led us to investigate broader aspects of skin appendage formation in two ways. To understand better the the repertoire of gene involved in the overall process of skin appendage formation, we have isolated keratinocytes from both Eda-positive and Eda-ablated mice, and are carrying out gene expression analyses to assess active signaling pathways that are dependent or independent of Eda action. A subset of those genes are expected to act in sebaceous gland formation;and in parallel, we focus on "trophic" (growth) effects of Eda in sebaceous glands by examining in detail the genes expressed specifically during sebaceous gland formation and in the sebocytes that populate them;and determining the effects of graded levels of Eda on the processes of growth and development. To identify EDA target genes, we have done the expression profiling to infer genes differentially expressed at various developmental time points in primary keratinocyte cultures established from wild-type and Tabby embryonic skin. By gene expression profiling, we found quite different cohort of genes significantly affected in Tabby keratinocytes, including Tbx1, Bmp7 and Jag1. Their functions under Eda regulation become targets for further study. In the past year, we have focused on the regulation of the critical partner for Eda in all cases where it is known to act, the Eda receptor Edar. Transfection experments with a candidate promoter region and a reporter gene demonstrated that receptor expression responds to the important Wnt signaling pathway at two indispensible sites in the promoter DNA, and the Wnt system and Edar levels will now be studied in sebocytes and keratinocytes to look for variations that correlate with trophic effects.